Localization of plasmin on macrophages and activation of pro-MMP-9 play key roles in macrophage recruitment in the inflammatory response. These functions are promoted by plasminogen receptors exposing C-terminal basic residues on the macrophage surface. Recently, we identified a novel transmembrane plasminogen receptor, Plg-R KT , which exposes a C-terminal lysine on the cell surface. In the present study, we investigated the role of Plg-R KT in macrophage invasion, chemotactic migration, and recruitment. Plg-R KT was prominently expressed in membranes of human peripheral blood monocytes and monocytoid cells. Plasminogen activation by urokinasetype plasminogen activator (uPA) was markedly inhibited (by 39%) by treatment with anti-Plg-R KT mAb. Treatment of monocytes with anti-Plg-R KT mAb substantially inhibited invasion through the representative matrix, Matrigel, in response to MCP-1 (by 54% compared with isotype control). Furthermore, chemotactic migration was also inhibited by treatment with anti-Plg-R KT mAb (by 64%). In a mouse model of thioglycollate-induced peritonitis, anti-Plg-R KT mAb markedly inhibited macrophage recruitment (by 58%), concomitant with a reduction in pro-MMP-9 activation in the inflamed peritoneum. Treatment with anti-Plg-R KT mAb did not further reduce the low level of macrophage recruitment in plasminogennull mice. We conclude that Plg-R KT IntroductionActivation of plasminogen, the zymogen of the primary thrombolytic enzyme plasmin, is markedly promoted when plasminogen is bound to cell surfaces (for review, see Miles et al 1 ) and cellassociated plasmin is protected from inactivation. 2,3 Therefore, cells become armed with the broad-spectrum proteolytic activity of plasmin. 4 This provides a mechanism to facilitate both physiologic and pathologic processes requiring cell migration. Plasminogendependent cell migration is involved in macrophage recruitment during the inflammatory response, 4-10 tissue remodeling, 11 wound healing, 12,13 tumor cell invasion and metastasis, 14,15 skeletal myogenesis, 16 neuroendocrine prohormone processing, 17,18 and neurite outgrowth. 19,20 Studies in plasminogen-deficient mice have demonstrated that plasminogen plays a key role in cell migration in a diverse array of physiologic and pathophysiologic settings, notably, macrophage recruitment in response to inflammatory stimuli in the thioglycollate-induced model of peritonitis. Plasmin-dependent cell migration is accomplished by direct degradation of extracellular matrix components by plasmin and also by activation of matrix metalloproteinases for further degradation of extracellular matrices. [4][5][6][7] Among the plasminogen-binding proteins, those exposing Cterminal basic residues on cell surfaces are predominantly responsible for the ability of eukaryotic cells to enhance plasminogen activation, because carboxypeptidase B (CpB) treatment abrogates cell surface-dependent plasminogen activation. 21 Furthermore, plasminogen-dependent macrophage recruitment is mediated by CpB-sensitive plasminoge...

Thrombomodulin is an essential cofactor for the activation of the anticoagulant protein C by thrombin. We have identified the expression of thrombomodulin messenger RNA (mRNA) and protein in peripheral blood monocytes. While untreated monocytes expressed thrombomodulin mRNA by Northern blot analysis, lipopolysaccharide-treated cells had decreased mRNA expression. Thrombomodulin antigen was shown in the cytoplasm and on the surface of monocytes by immunohistochemical staining, and thrombomodulin activity was shown on the surface of intact monocytes. One population of synovial lining cells that normally expressed mononuclear phagocyte antigens also expressed thrombomodulin in both noninflamed osteoarthritic synovium and in inflamed rheumatoid arthritis synovium. However, these cells did not express another endothelial protein, von Willebrand factor. We conclude that both circulating and tissue mononuclear phagocytes are capable of expressing thrombomodulin.

When plasminogen binds to cells its activation to plasmin is markedly enhanced compared to the reaction in solution. Thus, cells become armed with the broad spectrum proteolytic activity of plasmin. Cell-surface plasmin plays a key role in macrophage recruitment during the inflammatory response. Proteins exposing basic residues on the cell surface promote plasminogen activation on eukaryotic cells. We have used a proteomics approach combining targeted proteolysis with carboxypeptidase B and multidimensional protein identification technology, MudPIT, and a monocyte progenitor cell line to identify a novel transmembrane protein, the plasminogen receptor, Plg-RKT. Plg-RKT exposes a C-terminal lysine on the cell surface in an orientation to bind plasminogen and promote plasminogen activation. Here we review the characteristics of this new protein, with regard to membrane topology, conservation of sequence across species, the role of its C-terminus in plasminogen binding, its function in plasminogen activation, cell migration, and its role in macrophage recruitment in the inflammatory response.

Plasminogen activation is markedly enhanced on cell surfaces. Cell‐bound plasmin plays a key role in cell migration. Plg‐RKT is a novel transmembrane plasminogen receptor induced when promonocytes differentiate. Here we investigated the role of Plg‐RKT in macrophage migration using an anti‐Plg‐RKT mAb that competes with plasminogen for binding to cells. Chemotaxis was stimulated by MCP‐1 in transwells using U937 monocytoid cells that expressed high levels of Plg‐RKT. In the presence of plasminogen, cell migration was markedly decreased by treatment with anti‐Plg‐RKT mAb compared to treatment with isotype control. In a peritoneal inflammation model, C57BL/6 female mice were injected intravenously with either anti‐Plg‐RKT mAb or isotype control prior to intraperitoneal injection with 3% thioglycollate, followed by a second intravenous injection of mAb. Cells were harvested by peritoneal lavage. Anti‐Plg‐RKT mAb inhibited macrophage recruitment in a dose‐dependent manner [at the maximum dose of 27 μg/g, 58 ± 5.4 % fewer macrophages were recovered compared to the isotype control (p <0.001, n=5)]. Macrophage recruitment was significantly decreased in plasminogen null mice compared to wild type littermates, as reported. We found no effect of anti‐Plg‐RKT mAb upon macrophage recruitment in plasminogen null mice. These results suggest a major role of Plg‐RKT in the regulation of the inflammatory response.